EP0191850A1

EP0191850A1 - Differentiation, identification and enumeration of neuthrophils, erythroblast and sub-populations of lymphopcytes with basic blue 141

Info

Publication number: EP0191850A1
Application number: EP19850904434
Authority: EP
Inventors: Lawrence Kass
Original assignee: Individual
Current assignee: Individual
Priority date: 1984-08-09
Filing date: 1985-07-27
Publication date: 1986-08-27
Also published as: WO1986001293A1

Abstract

La différentiation, l'identification et l'énumération de trois types de cellules de sang humain avec le colorant bleu basique 141. Dans un type, seuls les neutrophiles mûrs parmi tous les leucocytes se teintent d'un rouge-violet vif, alors que les leucocytes immatures de tous les types se teintent de bleu pâle. Dans un deuxième type, les érythroblastes tant mûrs qu'immatures se distinguent du premier type, les uns par rapport aux autres, et de toutes les autres cellules connues susceptibles de se rencontrer dans des échantillons de biopsie humaine par un spectre différent de couleur, c'est-à-dire, une couleur cuivrée distinctive. Dans un troisième type, les diverses sous-populations de lymphocytes (cellules B, cellules T helper, cellules T de suppression et cellules tueur naturelles) se dintinguent les unes des autres par des différences caractéristiques de grandeur, de teinte et de chromaticité.The differentiation, identification and enumeration of three types of human blood cells with the basic blue dye 141. In one type, only the mature neutrophils among all leukocytes stain a bright red-purple, while the Immature leukocytes of all types are colored pale blue. In a second type, both mature and immature erythroblasts are distinguished from the first type, relative to each other, and from all other known cells likely to be encountered in human biopsy samples by a different color spectrum, c that is, a distinctive copper color. In a third type, the various lymphocyte subpopulations (B cells, helper T cells, suppression T cells and natural killer cells) are distinguished from one another by characteristic differences in size, shade and chromaticity.

Description

DIFFERENTIATION, IDENTIFICATION AND ENUMERATION OF NEUTHROPHILS, ERYTHROBLASTS AND SUB-PUPULATIONS OF LYMPHOCYTES WITH BASIC BLUE 141

FIELD OF THE INVENTION The invention herein disclosed is based upon the discovery of a single dyestuff which has a staining function specific to three classes of human blood cells and which identifies, differentiates and makes possible enumeration of each of the classes. In one class, mature neutrophils alone of all leukocytes are stained a bright red-violet color, while immature leukocytes of all types stain pale blue. In a second class, both mature and immature erythroblasts are distinguished from the first class as well as from each other and all other known cells that might be present in human biopsy specimens by a different color spectra, namely, a distinctive copper color. In a third class, the various sub-populations of lymphocytes (B cell, T helper cell, T suppressor cell and Natural Killer cell) can be distinguished from one another on the basis of distinctive differences in size, hue and chroma.

DESCRIPTION OF THE PRIOR ART Historically, dyes have been used both alone and in various combinations and permutations to stain biological specimens to reveal the intricate structures of the specimen and sub-parts thereof for general studies, identification and differentiation as well as enumeration. Staining of human biopsy specimens from various parts of the body including, as illustrative; blood, bone, marrow and the various body fluids, including spinal fluid, urine, pus, secretions from various organs, etc., for information pertinent to human well-being is an established technique.

Before staining with a selected dye or combination of dyes, pre-treatment of the specimen has led to the development of the fields of cytology and cytochemistry which are adjunct areas of background in the present advance in the related arts. Before staining in some instances the sample of human matter is subjected to extensive chemical treatment. In a large number of instances the histological specimen after biopsy or removal from a selected organ or function, is fixed. Fixatives are generally preservatives and denaturants and may be essential to the dye function. In some instances fixatives interfere completely with the reaction and co-reaction of the dye with the biochemistry of the specimen. Cytochemistry includes considerable information on techniques developed to assure reproducible optical response in diagnostic examinations. Dye staining serves as a means of discernment of otherwise indiscernable detail by distinctive color reactions on cells and their associated stainable components; metabolic; functional and/or pathological. Research work involving microscopically small cells originally has been accomplished primarily by the use of the microscope in conjunction with selected energy sources including both white and fluorescent light. More recently, however, the monochromatic light made available by the development of lasers has increased. the scope of means for examination of variously dyed biological specimens for medically related ends and has brought advanced high technology means into existance and availability.

Among the various kinds of leukocytes found in blood, neutrophils have been known for many years as important factors in inflammatory response. For example, increased numbers of neutrophils are found in areas of bacterial inflammation, such as pus or infected urine. Conversely, decreased numbers of neutrophils in the blood leads to an increased risk of infections. Various studies have demonstrated that neutrophils actively phagocytize foreign particles, including bacteria, and that the granules in neutrophils contain enzymes that are capable of destroying microorganisms. Clinically, the laboratory technique often first used to detect infection in a suspected abcess or in the urine prior to specific microbiological verification and identification of an invading organism is the determination of increased numbers of neutrophils in a biopsy specimen of fluid, or tissues from affected areas of the body.

Prior art discloses a number of ways of identifying increased numbers of neutrophils in a specimen. Using standard Romanowsky type staining mixtures, neutrophils can be identified on fixed stained smears of the fluid or tissue in question. However, other types of blood cells, such as eosinophils, lymphocytes, monocytes and basophils are also stained by these types of panoptic reagents. Another method involves the direct examination of an unstained wet mount under the microscope, and presumptive identification of neutrophils in a specimen by means of their shape, size and nuclear configuration. However, identification of these cells in an unstained specimen is subject to inaccuracies, particularly mis-identification of neutrophils as other types of leukocytes.

Additional methods to circumvent these problems have involved the use of cytochemical stains. Using a stain for myeloperoxidase activity, neutrophils can be identified in specimens as a result of their distinctive staining properties of their granules. However, other types of immature neutrophils, such as promyelocytes, myelocytes, metamyelocytes and bands usually contain perioxidase activity as well, so the stain is not selective for neutrophils alone. Moreover, the test for myeloperoxidase requires fixation, and as it is usually performed requires two or more cytochemical reagents. Some of these (benzidines, etc.) have been implicated as possible carcinogens. Neutrophils are also identified in urine specimens by the use of supravital stains as formulated by Stemheimer and Malbin using crystal violet acetate and safranin which characteristically stain the granules and nuclei of these cells. However, these dyes in combination as formulated are not specific only to mature neutrophils, since, other developmental stages of neutrophilic granulocytes, as well as othr kinds of leukocytes and nonhematopoietic particles, like casts, are stained by these dyes. Stain selectivity is lacking and specific neutrophil enumeration can be difficult. With naphthol ASD-chloroacetate as the substrate and a variety of azo dyes as couplers, a specific esterase can be demonstrated in the granules of neutrophils in fixed preparations. As in the case of myeloperoxidase, specific esterase is not found exclusively in neutrophils, since it is detected in substantial amounts in all other developmental states of neutrophilic leukocytes including promyelocytes, myelocytes, metamyelocytes and bands. A recent adaptation of the specific esterase method measures release of indoxyl from indoxyl carbonic acid ester by esterases of neutrophils in a specimen of urine. Indoxyl is oxidized by atmospheric oxygen to indigo blue. Using this reaction, a rapid test for the presence of neutrophils in a urine specimen has been developed and is being used in the clinical laboratory.

As best can be determined from a thorough search of the prior art no other methods are revealed for the direct, rapid differentiation, identification and enumeration of neutrophils alone without cytochemical use of synthetic substrates.

Basic blue 141 (also known as Basacryl Blue X-7GFL) is also useful in similar identifications of erythrocytes which have no nucleus, and their precursor erythroblasts including proerythroblasts and intermediate normoblasts both of which have coppery-red nuclei and blue cytoplasm, the normoblasts of smaller area (or volume). More recently it has also been found further cell identifications can be differentiated after formalin-acetone-acetic acid (FAA) fixation (as is also necessary with the above erythroblasts) in the case of the subclasses of lymphocytes including B cells, T helper cells, T suppressor cells and Natural Killer (NK) cells. Plate II illustrates a black and white concept of these cells which can here be verbally described only in association with the Plate as to approximate and descriptive color of the graphic identifying cell pattern. An extensive computer data base and literature search with respect to basic blue dye 141 does not suggest uses for this dyestuff other than as a textile dye except in a published paper dealing with this dye along with several hundred other dyes in wastewater sewage treatment. No suggestion has been found in the literature as to any properties and uses of basic blue 141 as a specific stain for neutrophils, or for mature and immature erythroblasts, or for the various subpopulations of lymphocytes. DETAILED DESCRIPTION OF THE INVENTION This invention introduces and teaches direct and selective staining of neutrophils, erythroblasts, and sub-populations of lymphocytes through the use of one specific dyestuff, namely, basic blue 141. Basic blue 141 is a basic, cationic dye containing an oxaziήe chroraophoric group. It is listed in the Colour Index as a textile dye, but its chemical structure has not been specifically shown. From an extended experience with various lots of the dye, basic blue 141, some variations in response has been noted. Some lots are useful directly in aqueous solutions. Where response has been at variation, adjustment of the pH level of the lot to between 5.2 to 5.5 has been corrective. Plate I identifies the compound on the basis of its characteristic spectrophotometric pattern, and is relied upon as the "finger print" of the sonamed compound.

In practice of the invention, a 1% aqueous distilled water solution of the powdered dye is prepared and filtered. In use, the aqueous solution of the dye is applied directly to the surface of the glass slides coated with blood, buffy coat, pus, urine sediment or other biopsy fluids or tissues containing blood cells including bone marrow aspirated from the patient. Alternatively, a staining jar can be used. Although the stain is effective using partially denatured biopsy specimens that have been fixed for five minutes in absolute methanol or FAA fixative, coloration of the neutrophils is particularly intense and specific using dried, unfixed specimens.

The cationic dye may also act as a fixative, since the cells do not wash off a prepared slide to any substantial degree after staining and drying. Upon flooding the biopsy coated surface area of the glass slide with the 1% aqueous solution of the dye stain, a golden sheen develops on the surface of the liquid film on the slide after about 30 seconds exposure. Where the biopsy coating on the slides contains primarily neutrophils, the golden sheen spreads evenly over the surface of the aqueous liquid dye surface. Where the biopsy coating on the slides contains primarily lymphocytes, the sheen may appear particulate and dispersed. After an effective time interval of contact of about five minutes, the slides are washed in distilled water until no further bleeding of the dye into the water is detected. The slides are air-dried and further examined under white light absorbance under the light microscope. Coverslips should be mounted on the slide with the use of immersion oil rather than mounting media containing xylene (e.g., Permount), since the reaction product in neutrophils dissolves rapidly in these materials containing organic fixatives, and eventually disappears. Using the direct glass slide or coverslip method, granules of neutrophils stain bright reddish-purple. Nuclei of neutrophils show a purple outline, and pale blue staining of nuclear chromatin. Granules and cytoplasm of other leukocytes, such as eosinophils, basophils and monocytes stain pale blue as do platelets. Brightly staining red purple granules as observed in the case of neutrophils are not found among any of the other leukocytes. In bone marrow specimens, the nuclei and cytoplasm of promyelcytes, myelocytes, metamyelocytes and bands stain pale blue. In the foregoing laboratory work, the red purple granules of the type consistently found in mature neutrophils could not be characteristically identified in the immature leukocytes including those immature cells of the neutrophilic, basophilic or eosinophils series.

Of additional interest, in methanol-fixed or FAA-fixed specimens of bone marrow, nuclear chromatin of mature and immature erythroblasts (proerythroblasts and intermediate normoblasts) stained a distinctive bright copper color, with exceptionally clear delineation of the nuclear membranes and nuclear chromatin pattern. Nuclei of all other marrow cells stained pale blue. Mature erythrocytes and cytoplasm of mature erythroblasts stained a grayish-copper color, whereas the cytoplasm of proerythroblasts stained deep blue. Used in this fashion, erythroblasts at all stages of maturation were made immediately visible in bone marrow specimens, facilitating their quantitation in the process of bone marrow Interpretation.

When the dye was used as a dilute supravital stain with iving blood cells, all leukocytes stained pale green, and no onfident distinctions between neutrophils and other leukocytes resent could be made on the basis of differential and Lstinctive coloration of granules.

As an adaptation of the glass slide staining technique, forts were made to develop a "spot" test for the presence neutrophils in a body fluid or other biopsy specimen, ing Whatman #1 filter paper, spots of the following were de on the paper and evaporated to dryness at room temperaees: 1) hemolysate, normal blood 2) red blood cells plus normal plasma 3) buffy coat leukocytes containing approximately 80% neutrophils, 15% lymphocytes, 3% monocytes and 2% eosinophils 4) cell suspensions containing 90% lymphocytes, 5% neutrophils and 5% monocytes. Table I below demonstrates the color reaction that occured when a 0.1% aqueous filtered solution of the dye was applied to the spot, after 1 minute and after 6 hours:

In a second type of "spot" test, a drop of 1% aqueous filtered solution of basic blue 141 was applied to a piece of Whatman #1 filter paper, and air dried at room temperature. With this technique, a round, blue colored spot of filter paper impregnated with basic blue 141 was formed, and measured approximately 1.0 cm in diameter. As shown in Table II, the following color reactions were seen when a drop of fluid of differing types was placed in the center of the blue spot:

In the case of buffy coat containing many neutrophils and urine containing many neutrophils, the center of the spot remained light gray or pale blue, but after several minutes, presumably after diffusion of the reaction product to the edges of the spot, the spot was ringed by a thin, bright red-violet colored rim. After 4-5 minutes, this redviolet color became dark purple.

In dry powder form, the shelf life of the dye basic blue 141 is indefinite. In diluted aqueous form, the staining qualities of the dye remain stable and unaffected at normal room temperatures for at least one year of shelf life. Attention is directed to Plate I showing the spectral response curve of basic blue 141, identified sometimes under the name of Basacryl Blue X-7GFL. (This dye is available and may be supplied through BASF-Wyandotte U.S.A.) The curve of Plate I is relied upon for identification of the essential dye in the absence of well-established, known chemical structure. Selective and specific staining of neutrophils and erythroblasts by basic blue 141 is useful in medical diagnosis because:

1. Basic blue 141 quickly and specifically detects the presence of neutrophils, or inflammatory cells, in specimens, making the diagnosis of infection more rapid than before.

2. Basic blue 141 complements traditional panoptic and cytochemical stains for granulocytic cells by being specific for neutrophils alone among leukocytes, thereby permitting their quantitation in specimens.

3. Basic blue 141 also stains both mature and immature erythroblasts in bςne marrow specimens selectively and distinctively, thereby permitting precise quantitation of erythropoiesis in bone marrow interpretation and in the dis orders of erythropoiesis, illustratively anemia.

4. Basic blue 141 also facilitates identification of early erythroblasts (proerythroblasts) which can sometimes be difficult to identify using traditional panoptic stains; medically, identification of proerythroblasts may be important in the diagnosis of various anemias and leukemias.

Selective and specific staining of neutrophils by basic blue 141 advances the art in the following particulars:

1. Basic blue 141 requires only one dyestuff, thereby circumventing problems involved in dye mixtures and differing lots of the same dyes.

2. Basic blue 141 does not require addition of a synthetic substrate, as in the peroxidase or specific esterase reactions. 3. Basic blue 141 does not require prior fixation. It is functional on dried but non-fixed preparations, and on methanol-fixed and FAA-fixed preparations. It is not effective as a supravital stain for neutrophils alone. 4. Basic blue 141 can be adapted as a "spot" technique, utilizing a rapid color change to red-violet as the reaction product or end point to identify the presence of neutrophils in the specimen. This is accomplished by use of an inert but integral porous matrix impregnated with the dyestuff to produce a one use, throw-away test unit. This impregnated test unit, wet or dry, can be applied to a suspected specimen or dipped into a specimen in an aqueous environment, allowing leukocytes to adhere to it and interact with the dyestuff to produce a highly and characteristially colored reaction product when neutrophils are prevalent.

Illustrative matrix material for surface coating or impregnation to produce a carrier for the basic blue 141 dye suitable for "spot" identification technique include as illustrative, but not limiting, shaped disks or rods of porous paper (filter paper) or water-wettable porous plastic forms suitable for (A) first wetting with a small retained amount of the dye and at a later time, if desired, re-wetting with the human biopsy specimen in a suitable (aqueous) environment for color change and neutrophil identification. A surprising further specialized use of basic blue 141, more recently discovered, is the utility of the dyestuff in the identification of subclasses of lymphocytes including B cells, T helper cells, T suppressor cells and Natural Killer (NK) cells with remarkable individual differentiation capability. A presently developed technique found useful is to fix coverslip films of peripheral blood or bone marrow or alternatively blood smears in FAA fixative (95% ethanol - 90 ml; glacial acetic acid - 5 ml; 37% formalin solution - 5 ml). Time - 5 minutes. Wash the films in distilled water.

The so prepared slide is flooded with the dye solution in distilled water made by adjusting a 1% solution of the basic blue 141 dye to a pH of 5.2 to 5.5 by use of the Trizmal (Tris maleate) pH 7.6 buffer concentrate, 200 mM/ liter. At the specified pH, a golden sheen forms on the surface of the aqueous dye solution and a color change from a blue to a blue violet is observed. Stain for about five minutes. Excess dye is removed by washing the slide with distilled water; blot dry on bibulous paper. Mount the coverslip with immersion oil for use under conventional light microscopy.

In the original work using normal heparinized peripheral venous blood and Ficoll-Hypaque gradient preparations from normal individuals, the various lymphocyte sub-populations were separated with 95% purity or greater in a commercial cell sorter. The test cell populations were originally identified by means of the following antibodies: B cells by the F(ab)2 antibodies, T helper cells by the OKT4 antibody, T suppressor cells by the OKT8 antibody and Natural Killer (NK) cells by the leu 7 antibody. After the individual cell populations were separated into individual collections, the separated populations were concentrated by cytocentrifugation and deposited on a cleaned glass coverslip. The coverslips were individually fixed in FAA fixative and stained with the aqueous basic blue 141 dye as described above.

By the foregoing procedure, the staining characteristic of each of the above identified cells in a highly purified state could be observed, as shown in Plate II. B cells are identified by their pale green nucleus, colorless to faint blue cytoplasm and absence of granules. T helper cells are distinguished by their deep blue green nucleus, very dark blue scant cytoplasm, absence of granules, but dark blue "speckles" in the nucleus. T suppressor cells have a blue-green nucleus and a deep blue scant cytoplasm containing a plurality (to about 6) of tiny red granules in the cytoplasm. The Natural

Killer (NK) cells are larger than all of the foregoing cells and are characterized further by a very pale green nucleus and a colorless to faint blue abundant cytoplasm containing many large red granules. The T suppressor and T helper cells are clearly smaller than the B-cells and the NK cells.

Selective and specific staining of the lymphocyte subpopulation by basic blue 141, as above, has the following advantages over the known prior art and currently available techniques:

1. The biopsy specimen need not be freshly secured, may be dried out at toom temperatures and still provide viable and characteristic color distinctions. 2. The procedure is unusually rapid, compared to the more tedious immunoiogical methods that may take substantial specimen preparation.

3. The colors are uniquely vivid with respect to color reaction and differences in color. In essence, the various subpopulations of lymphocytes (B, T helper, T suppressor and Natural Killer) are "color coded".

4. Color differentiation and enumeration can be utilized in a differential cell count, since all of the other formed elements of the blood can also be identified. In complex iramunological techniques, usually only one or two cells types can be identified.

5. The method can be used for the rapid diagnosis of the immunologic subtype of acute lymphoblastic leukemia or chronic lymphocytic leukemia, prior to the institution of specific therapy. In the case of the T cell malignancies, identification of this particular subtype may have important prognostic implications.

6. Rapid identification of lymphocyte subpopulations is made possible in lymph node imprints, particularly in the distinction between reactive hyperplasia in which T and B cells may be found in conjunction with each other, and malignant lymphoma in which all of the cells are either B or T cell type. As above, identification of these cells with a simple staining technique provides many advantages, including immediacy and inexpensiveness, compared to more complex immunologic methods presently known.

7. Costly monoclonal antibody reagents are not needed.

8. Permits a rapid microscopic screening technique for determination of the percentage of the various lymphocyte subpopulations in many other conditions, such as; malignancies, Hodgkin's disease, collagen disease, AIDS and systemic lupus erythrematosis. EXAMPLES

The following Examples are illustrative of the utility of the invention in diagnostic end uses:

1. A 58 year old man developed pain and tenderness in the inguinal area. On physical examination, a tender red mass measuring 5.8 cm. in the right groin was found. The mass was aspirated with a needle and syringe. One part of the sample was submitted for bacteriologic cultures, another part was smeared onto a glass slide, quickly air dried, and stained without fixation with a 1% aqueous solution of basic blue 141. Microscopic analysis revealed striking and unusual numbers of neutrophils identified by characteristic redpurple stained granulation.

2. A 73 year old woman complained of burning upon urination. On physical examination, her temperature was 102 degrees F., and the lower abdominal area was tender. One part of a urinalysis was obtained and submitted for culture. A spun (2000 R.P.M. for five minutes) sediment of a second part of the urine sample was spotted on filter paper, quickly air dried and stained with an aqueous 1% solution of basic blue 141. Within one minute, the spot turned red purple.

Strikingly increased numbers of leukocytes present in the urine was confirmed by supravital staining of the sediment with cresyl violet and safranin as a check.

3. A 52 year old man was evaluated for weakness and fatigue. He was found to have an enlarged spleen. Laboratory tests revealed hemoglobin 7 grams %, hematocrit 22%, white blood cell count 4200/mm³ with normal differential, and platelets 143,000/mm³. A bone marrow specimen was [obtained. A part was stained with Wright's stain. Simultaneously, a methanolfixed coverslip containing a part of the bone marrow specimen was stained with basic blue 141. Strikingly increased numbers of mature and immature erythroblasts, along with neutrophils, were stained differentially and selectively by this dyestuff, permitting their quantitation with ease.

4. A 3 year old child was brought to the emergency room with a history of fever and lethargy. On physical examination, the child was semi-comatose and had a grand mal seizure. A spinal tap was performed. The fluid was cloudy. After a first aliquot sample of the spinal fluid was sent for bacteriologic analysis and Gram stain, a dried, unfixed cytocentrifuged second aliquot portion of the sample was stained with basic blue 141. Large numbers of neutrophils were found,, consistent with bacterial infection as subsequently detected and reported on the bacterial analysis.

5. A 49 year old white male was admitted to the hospital for evaluation of splenomegaly. On physical examination, blood pressure 110/72, pulse 80/minute and he was afebrile.

Enlarged lymph nodes could be palpated in the cervical and axillary areas, and the spleen was markedly enlarged. Laboratory values revealed hemoglobin 10 gm%, white blood count 230,000/mm³, and platelet count 87,000/mm³. On Wright's stain of the peripheral blood, nearly all of the cells were intermediate sized lymphocytes. Immunologically using immunoperoxidase techniques, the lymphocytes were B cells. On a separate slide stained with basic blue 141, all of the lymphocytes had pale green nuclei, and cytoplasm that was virtually unstained or faint blue, characteristic of B lymphocytes. The diagnosis was chronic lymphocytic leukemia.

6. A 3 year old black female child was admitted to the hospital because of irritability and intermittent fever. On physical examination, blood pressure 100/82, pulse 120/ minute, and temperature 39.5ºC. Enlarged axillary lymph nodes and enlarged liver and spleen were noted. Laboratory values disclosed hemoglobin 7 gm%, white blood count 54,000/ mm³, and platelet count 32,000/mm³. On Wright's stained smear of the peripheral blood, most of the cells were lymphoblasts. Cytochemically, these lymphoblasts contained focal unipolar activity of acid phosphates, a marker for T cells. Immunologically, using monoclonal antibodies, the leukemic lymphoblasts were T cells. Using basic blue 141, the lymphoblasts showed dark, blue-green nuclear staining and deep blue cytoplasmic staining, characteristially found in identification of T helper cells with this dye. The diagnosis was acute lymphoblastic leukemia, T cell convoluted type. Accompanying Plate II details black and white sketches of the enlarged cells, pointing out verbally color descriptors which are guides to the missing color reproductions. Size relationships and color descriptors are useful, but not mathematically precise. Use of the modifier "denatured" as is used in the appended claims embraces in the most mild form a drying out of the biopsy specimen before treating with the aqueous dye solution, a second degree of denaturation is the further treatment with the dye solution after drying and a third degree of denaturation is a treatment of the biopsy sample with FAA or equivalent fixative of the prior art before subjecting the leukocytes present to the aqueous dye basic blue 141 solution. In the case of analysis of the subspecies of lymphocytes as disclosed herein, use of the fixative pre-treatment is a preferred mode of procedure. Having illustrated the best mode of practice of my invention presently known to me, what is claimed is:

Claims

1. A composition of matter comprising the reaction product of a human biopsy specimen containing denatured blood cell leukocytes suspended in an aqueous environment with which the dye basic blue 141 is reacted therewith to identify and differentiate neutrophils, immature erythroblasts and the sub-populations of lymphocytes characteristically selectively reactive when present in the admixture.

2. The composition of Claim 1, wherein the aqueous environment is optimally within a range of pH from about 5.2 to 5.5.

3. The composition of Claim 1 wherein the blood cell leukocytes include neutrophils.

4. The composition of Claim 1, wherein the blood cell leukocytes include immature and mature erythroblasts.

5. The composition of Claim 1, wherein the human biopsy cells include a subpopulation of pre-fixed lymphocytes identified by their individually differential color reactivity with said dye in said composition of pH of from about 5.2 to 5.5.

6. The composition of Claim 5, wherein the subpopulations of lymphocytes embrace B cells, T helper cells, T suppressor cells and Natural Killer (NK), cells.

7. The method of analysis of the compositions of Claim 2 which identifies, differentiates and permits means for enumeration of the neutrophils present through their characteristic bright red magenta granules and pale blue nucleus.

8. Method of analysis of the composition of Claim 4 which comprises identification, differentiation and permits means for enumeration of immature and mature erythroblasts by their distinctive coppery coloration of their nuclei, relatively dark blue surrounding cytoplasm and their relative dimensions.

9. The method of analysis of the composition of Claim 5,which comprises identification, differentiation and enumeration of B cells present therein by their pale green nucleus and colorless to faint blue cytoplasm free from granules; T helper cells by their deep blue-green nucleus, darker blue spots distributed therein and granule-free dark blue cytoplasm; T suppressor cells by their blue green nucleus, deep blue surrounding cytoplasm containing a single clustered plurality of tiny red granules not exceeding about ten in the cytoplasm; the Natural Killer (NK) cells by their very pale green nucleus, a cluster of numerous large red granules arrayed in a colorless to faint blue abundant cytoplasm and said cells being of relatively larger size than said foregoing B and T cells.

10. The method of analysis of the mature and immature erythroblasts and erythrocytes in the compositions of Claim 1 which comprises differentiating the erythrocytes present by their distinctive uniform copper colored solid areas, the colored nuclei and dark blue cytoplasm and the intermediate normoblasts by their similar color pattern but relatively small overall diameters.

11. An analytically useful device or article for use in identification of the class of cells consisting of mature neutrophils and erythrocytes present in a human biopsy diagnostic specimen which comprises a porous element characterized by the presence in at least a visible flat surface portion thereof of said element of a minor amount of the dye basic blue 141 within and about said dye responsive surface defining an active area of said porous element.

12. The analytical device or article of Claim 4 where the specific identification comprises red blood cells selected from the group consisting of mature and immature erythroblasts and erythrocytes.

13. The article of Claim 4, when the specific identification comprises mature neutrophils.

14. A composition of matter comprising the red-violet colored reaction product resulting from an intimate contact of the dye basic blue 141 with granules of neutrophils, identifying and differentiating mature neutrophils from all other cells present in a human biopsy specimen.